Shami, Annelie

Introduction: Co-stimulatory proteins are master regulators of the immune system. We identified the co-stimulatory CD40-CD40L dyad as a key driver of inflammation during atherosclerosis and have shown that CD40 and CD40L exert cell-type divergent functions. Hypothesis: CD40 is abundantly expressed on endothelial cells (EC) undergoing endothelial mesenchymal transition (EndMT). We hypothesize that EC CD40 drives atherosclerosis via EndMT. Methods: We analyzed scRNAseq data from human carotid plaques, performed multiplex IHC, and used human aortic endothelial cells (HAECs) for in vitro studies. A tamoxifen-inducible EC specific CD40 deficient atherosclerotic mouse model (cadherin5 (cdh5)ERT2-CD40 flfl -ApoE -/- ) was generated. Results: Besides its presence on immune cells, CD40 is expressed on endothelial cells in both human and mouse atherosclerotic plaques. Analysis of scRNAseq data of human carotid atherosclerotic plaques showed that CD40 is highly expressed in COL1A2/FN1 expressing endothelial cells (ECs), a subset undergoing EndMT. Multiplex staining revealed that CD40 colocalized with the EndMT markers αSMA, NOTCH3, TWIST and VCAM1 in human plaques. In vitro , stimulation of human arterial endothelial cells (HAECs) with CD40L resulted in an increase in the EndMT markers markers fibronectin, vimentin, sm22α, FSP1 and snail. Using phosphoproteomics, CD40L was found to decrease phosphorylation of the junctional integrity proteins CTNNA1 [S652; S655] and ARHGAP21 [S1383]. To investigate the endothelial-specific effects of CD40 in atherosclerosis, 8-week-old cdh5ERT2-CD40 flfl -ApoE -/- mice and their CD40 flfl -ApoE -/- littermate controls were treated with tamoxifen and fed a high cholesterol diet for 10 wks. Deletion of EC-CD40 resulted in a significant reduction in plaque size. This reduction was accompanied by a shift from fibrous cap atheromas (FCA) towards intimal xanthoma (IX) and pathological intimal thickening (PIT). Flow cytometry analysis of the spleen showed that CD40 deletion in ECs results in a significant decrease in CD8 and CD4 effector T cells. This decrease in CD8 and CD4 effector T cells was accompanied by a diminished activation status, shown by a decrease in CD69 + CD8 + CD62 - CD44 + T cells, CD40L + CD8 + CD62 - CD44 + T cells and CD40L + CD4 + CD62 - CD44 + T cells. These findings demonstrate that endothelial CD40 drives atherosclerosis by inducing EndMT-driven plaque inflammation.

Introduction: Atherosclerosis, the underlying cause of cardiovascular disease (CVD), is a lipid-driven chronic inflammatory disease. We have identified the co-stimulatory CD40-CD40L immune checkpoint dyad as a key driver of inflammation during atherosclerosis. CD40 and CD40L exert cell-type divergent functions via different signaling pathways. Hypothesis: We hypothesize that CD40 on endothelial cell (ECs) induces endothelial-to-mesenchymal transition (EndMT)-mediated vascular inflammation in atherosclerosis. Methods & Results: Using single cell RNAseq, we found that CD40 (TNFRSF5) is expressed on PDE3A + GJA5 + and KDR + COL15A1 + ECs of advanced human carotid atherosclerotic plaques. CD40 is highly expressed in the endothelial lining of human and mouse atherosclerotic plaques, and colocalizes with the EndMT markers fibronectin and collagen and adhesion molecule ICAM-1. CD40L activation of human aortic endothelial cells (HAECs) results in expression of the EndMT markers fibronectin, vimentin, sm22α, FSP1 and snail. Activation of CD40 in EC induces filamin A dependent translocation of CD40 into lipid rafts, increases TNF-receptor associated factor (TRAFs) -2, -3 and 6 levels, induces Akt signaling and increases expression of VCAM1 and CCL2. Conclusions: The endothelial cell has been more and more regarded as a cell type that plays a major role in driving inflammation. Our data suggest that the CD40-CD40L axis plays a significant role in EC-mediated inflammation in atherosclerosis by driving EndMT.

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

Abstract Aims CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice. Method and Results Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe−/− background were generated (CD40wt and CD40mac−/−, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac−/− compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac−/− atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b− macrophages in the atherosclerotic aorta of CD40mac−/− compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac−/− mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1). Conclusions We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.